Methods for diagnosis of low grade astrocytoma

ABSTRACT

A method for identifying low grade astrocytoma cells in a sample is provided, wherein the method distinguishes between low grade astrocytoma cells and normal astrocytes, thus permitting early diagnosis of astrocytoma. The method uses antibody directed against JI-31 to test astrocytes in the sample for the presence or absence of JI-31 polypeptide, the low grade astrocytoma cells being characterized by the absence of JI-31 while normal astrocytes are characterized by the presence of JI-31.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional application of U.S. patent applicationSer. No.10/269,773 filed Oct. 10, 2002, which claims the benefit of U.S.Provisional Application, Ser. No. 60/328,917, filed Oct. 11, 2001, thecontent of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The subject of the present invention is a method for using an antibodyto detect the presence of a disease, and more specifically todistinguish low grade astrocytomas from normal reactive astrocytes.

BACKGROUND OF THE INVENTION

Astrocytomas are members of the glioma family of tumors; that is, tumorsarising from glial cells. In the case of astrocytomas, the tumors arisefrom a type of glial cell called astrocytes.

Astrocytes, named by Spanish neuroanatomist Santiago Ramon y Cajal in1913 after he discovered the star-shaped cells, are the major cell typesof the central nervous system that responds to various pathologicalconditions, including trauma, ischemia, demylination, inflammation, etc.In response to such pathological conditions, the normally quiescentastrocyte becomes “reactive,” and may proliferate and migrate,exhibiting hypertrophy with increased expression of Glial FibrillaryAcidic Protein (GFAP), expression of cell surface MHC class I and IImolecules, and producing various cytokines and growth factors.

Neoplastic transformation of astrocytes give rise to a variety ofastrocytomas. Astrocytomas are generally grouped into two categories:low-grade (grades 1 and 2) and high-grade (grades 3 and 4). High-gradetumors grow rapidly and can easily spread throughout the brain, whilelow-grade astrocytomas are usually much more localized and grow slowlyover a longer period of time. The prognosis for patients with high-gradeastrocytomas is poor, with an average survival of 18 months for patientswith grade 3 astrocytoma (with radiation therapy and chemotherapy) andan average survival of from 17 weeks (no treatment) to 51 weeks (withsurgery and radiation treatment) for grade 4 astrocytomas.

Morphologically, low-grade astrocytomas are usually normal inappearance, making it difficult to distinguish low-grade astrocytomacells from normal quiescent or reactive cells. Some grade 2 low-gradeastrocytomas may be slightly abnormal in appearance, but are not easilydetected by routine microscopic evaluation of tissue biopsy samples. Bycontrast, the high-grade astrocytomas are abnormal in appearance andshow evidence of mitosis, making the cells easily identifiable bymicroscopic evaluation.

Visualization of astrocytes during microscopic evaluation of tissuesections is typically facilitated by staining the biopsy tissue samplewith antibody to GFAP, a cytoskeletal protein expressed both inquiescent and reactive astrocytes and in low grade and high gradeastrocytomas. Both monoclonal mouse and polyclonal rabbit anti-humanGFAP antibodies have been developed that are specific against GFAP, notrecognizing other intermediate filament proteins. These antibodiestypically react with both astrocytes and astrocytoma cells, and thus donot serve to differentiate between normal and transformed cells beyondfacilitating morphological inspection of the cells.

Although the prognosis for patients with grade 1 astrocytoma is good,with some patients known to live 30 years or more following diagnosis,the prognosis for patients with grade 2 low-grade astrocytoma is muchless optimistic. Recent studies have indicated that the 5-year survivalrate in grade 2 astrocytomas is about 34%. With radiation therapy, the5-year survival rate increased to about 70%. It is apparent that earlydiagnosis, and thus early treatment, is important in treating patientswith low-grade astrocytoma.

What is needed, therefore, is a reliable and efficient diagnosticprocedure for differentiating low grade astrocytoma cells from activeand quiescent normal astrocytes in tissue biopsies.

SUMMARY OF THE INVENTION

One aspect of the present invention is a method for identifying lowgrade astrocytoma cells in a sample containing astrocytes, comprisingtesting the astrocytes in the sample for the presence or absence ofJI-31 polypeptide, the low grade astrocytoma cells being characterizedby the absence of JI-31.

Another aspect of the present invention is a method for distinguishinglow grade astrocytoma cells from normal astrocytes, the methodcomprising the steps of:

providing a tissue sample from a site of a suspected astrocytoma;

contacting the sample with antibody molecules immunoreactive with JI-31protein, under conditions wherein the antibody molecules can form animmune complex with any JI-31 antigen in the sample;

detecting cells in the sample in which the immune complex is present,thereby identifying cells expressing the JI-31 antigen; and

detecting astrocytes present in the sample in which the immune complexis absent, thereby identifying low-grade astrocytoma cells.

Another aspect of the present invention is a method for identifying lowgrade astrocytoma cells in a sample, comprising the steps of:

contacting the biological sample with anti-JI-31 antibody underconditions in which the antibody binds to JI-31 antigen in the sample;

contacting the biological sample with a secondary antibody selected soas to form an immune complex with the anti-JI-31 antibody bound to JI-31antigen;

detecting cells expressing the JI-31 antigen by detecting the immunecomplex; and

detecting astrocytes in the sample in which the immune complex is notpresent, to thereby identify low-grade astrocytoma cells.

Another aspect of the present invention is a kit for in vitro diagnosisof low grade astrocytoma comprising anti-JI-31 antibody, a mediumsuitable for formation of an antigen-antibody complex, and reagents fordetection of the antigen-antibody complex.

Another aspect of the present invention is a kit for in vitro diagnosisof low grade astrocytoma comprising anti-JI-31 antibody and instructionsfor using anti-JI-31 antibody to distinguish between low gradeastrocytoma cells and other astrocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects and advantages of the presentinvention will become better understood with reference to the followingdescription, appended claims, and accompanying drawings wherein:

FIGS. 1A-1D show the histochemical localization of JI-31 antigen andGlial Fibrillary Acidic protein (GFAP) informalin-fixed/paraffin-embedded tissue sections. Mouse monoclonalanti-JI-31 antibody or anti-GFAP antibody shows strong reactivity withnormal astrocytes as shown by the arrow in FIGS. 1A (GFAP) and 1B(JI-31). The low grade (grade 2) astrocytoma cells showed no reactivitywith anti-JI-31 antibody (arrow, FIG. 1D); whereas the surroundingnormal astrocytes were positive (*, FIG. 1D). In contrast, anti-GFAPreacted with both the normal astrocytes and the grade 2 astrocytomacells (arrow, FIG. 1C). The tissue sections were counterstained withHarris hematoxylin (original magnification ×200).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of identifying low gradeastrocytoma cells and distinguishing such cells from normal, reactiveastrocytes, using anti-JI-31 antibody.

One aspect of the method of the instant invention for identifying lowgrade astrocytomas comprises immunostaining tissue biopsy samples withanti-JI-31 antibody to differentiate between normal, reactive astrocytesand low grade astrocytoma cells.

Another aspect of the method of the instant invention for identifyinglow grade astrocytomas comprises immunolabeling tissue biopsy sampleswith anti-JI-31 antibody for fluorescence microcopy to differentiatebetween normal, reactive astrocytes and low grade astrocytoma cells.

Still another aspect of the method of the instant invention foridentifying low grade astrocytomas comprises subjecting tissue biopsysamples to immunoelectron microscopy using anti-JI-31 antibody todifferentiate between normal, reactive astrocytes and low gradeastrocytoma cells.

Still another aspect of the method of the instant invention foridentifying low grade astrocytoma cells comprises extracting proteinsfrom tissue biopsy samples and immunoblotting with anti-JI-31 antibodyto differentiate between normal, reactive astrocytes and low gradeastrocytoma cells.

Still another aspect of the method of the instant invention comprisestesting cultured cells for the presence of low grade astrocytoma cellsusing anti-JI-31 antibody.

Still another aspect of the present invention comprises kits useful fordiagnosing low grade astrocytomas.

JI-31. JI-31 is a cytoskeleton-associated polypeptide, which has beenshown to be a 30-kD protein, distinct from GFAP and vimentin proteinsalso found in glial cells. By contrast, GFAP is a major cytoskeletonprotein.

The JI-31 polypeptide is expressed in both high grade astrocytoma cellsand normal astrocytes, with enhanced expression demonstrated in reactiveastrocytes following injury to the central nervous system (CNS). See,Singh, et al., (1986) Bioscience Reports 6: 73-80; Predy, et al., (1987)Bioscience Reports 7: 491-502; Predy, et al., (1988) J. of NeuroscienceResearch 19: 397-404; Malhotra, et al., (1989) J. of NeuroscienceResearch 22: 36-49; Singh, et al., (1992) Dendron 1: 91-108; Malhotra,et al., (1993) Brain Research Bulletin 30: 395-404; and Singh, et al.,(1994) Biomedical Letters 50: 163-172, all hereby incorporated byreference in their entireties.

Surprisingly, however, the present invention demonstrates that JI-31does not appear to be expressed in low grade astrocytoma cells. Thus,JI-31 is useful as a diagnostic tool for distinguishing low gradeastrocytoma cells from quiescent and reactive astrocytes, as well asfrom high grade astrocytomas.

Antibodies. The present invention contemplates the use of polyclonal andmonoclonal antibodies, including recombinant single-chain or otherantibody derivatives or fragments, against JI-31 polypeptide andfragments thereof.

The present invention also contemplates the use of polyclonal andmonoclonal antibodies against variants of JI-31 polypeptides, includingnaturally occurring allelic variants.

As used herein, unless specified to the contrary, “anti-JI-31 antibody”shall refer to a polyclonal or monoclonal antibody, includingrecombinant single-chain or other antibody derivatives or fragments,directed against JI-31 polypeptide(s) or fragments or variants thereof.

Methods of producing polyclonal antibodies are well known to thoseskilled in the art. Exemplary protocols which may be used are describedfor example in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, (JohnWiley & Sons, Inc, 1991) which is incorporated herein by reference, andAusubel et al., (1994-1998, supra), in particular Section III of Chapter11.

Alternatively, monoclonal antibodies may be produced using the standardmethod as, for example, described in an article by Kohler and Milstein(1975, Nature 256,495497) which is herein incorporated by reference.

Anti-JI-31 Monoclonal Antibody. One example of an anti-JI-31 monoclonalantibody was produced using tissue homogenate from the brain of amultiple sclerosis patient and hybridoma technology, as reported by S.K. Malhotra, et al., (1984) in Microbios Letters 26: 151-157, herebyincorporated by reference in its entirety. This anti-JI-31 monoclonalantibody is specific for astrocytes but recognizes different antigenicsite(s) than does anti-GFAP antibody. Specifically, this anti-JI-31monoclonal antibody reacts with the astrocytic cytoskeleton. The JI-31hybridoma cell line has been deposited with the ATCC as CRL-2253.

Detection of Low Grade Astrocytoma Cells. As discussed above, it hasbeen previously demonstrated that JI-31 polypeptide is expressed innormal astrocytes and in high grade astrocytomas, and may be detected inthose cells using an anti-JI-31 monoclonal antibody. The presentinventors have now found that JI-31 polypeptides are not detected in lowgrade astrocytomas, providing a new diagnostic method for identifyinglow grade astrocytoma cells, and thus facilitating earlier diagnosis andtreatment of patients with low grade astrocytoma.

In the practice of this invention, astrocytes are tested usinganti-JI-31 antibody, as described below. Optionally, the cells are alsostained with a non-cell specific stain, as with Harris hematoxylin, forexample, which stains cell nuclei a deep blue color, allowing all cellsto be easily visualized. Optionally, cells may also be stained withGFAP, to facilitate characterization of the morphology of theastrocyte/astrocytoma.

Following testing with the anti-JI-31 antibody, the astrocytes areexamined for the presence or absence of the JI-31 antigen and areidentified, as summarized below in Table 1. TABLE 1 MorphologicallyReacts with Anti-JI-31 Cell Type Abnormal Antibody Normal Astrocyte NoYes Reactive Astrocyte No Yes Low Grade Astrocytoma No No High GradeAstrocytoma Yes Yes

Thus, cells that appear as relatively morphologically normal astrocytes,but which do not contain JI-31 antigen, are identified as low gradeastrocytoma cells.

Methods for Detecting JI-31 in Cells. As will be appreciated by theskilled practitioner, there are several ways known in the art fordetecting the presence of a specific polypeptide in a cell. Some ofthese methods are discussed below. However, the present inventioncontemplates any technique whereby the presence or absence of JI-31 in acell, either cultured or in a tissue sample, may be determined.

Immunohistochemical Staining of Tissue Sample

Screening techniques such as Magnetic Resonance Imaging (MRI) are usedto identify potential brain tumor sites, and biopsy tissue, as from aneedle biopsy, is extracted from patients for analysis.

Such clinically obtained tissue samples are generally formalin fixed andparaffin embedded, although tissue samples may also be fresh/frozen.Both are suitable for use in the present invention forimmunohistochemical staining using an antibody directed to JI-31protein. The tissue samples are then examined microscopically for thepresence or absence of the JI-31 antigen in cells of interest. Theprocedure for preparing and staining these tissue samples is describedbelow in detail.

Prior to immunohistochemical staining, slides containing theformalin-fixed/paraffin-embedded tissue biopsy samples are warmedovernight at 60° C. Slides containing fixed tissue are incubated twicefor 5 minutes each in Histoclear, a clearing agent available fromNational Diagnostics, Atlanta, Ga., followed by two incubations of 3minutes each in absolute ethanol and two incubations of 3 minutes eachin 95% ethanol.

Slides containing formalin-fixed tissue samples that have been treatedas described above with Histoclear and ethanol, are incubated for 20minutes in a mixture of hydrogen peroxide and methanol. One suitableH₂O₂:methanol mixture is formed by adding 50 ml of 3% H₂O₂ to 200 mlmethanol.

The slides are next washed for 5 minutes in Delbecco phosphate bufferedsaline (DPBS). A 10× stock of DPBS is made by combining 11.5 g dibasicsodium phosphate (molecular weight [m.w.] 142.0), 2.0 g monobasicpotassium phosphate (m.w. 136.1), 2.0 g potassium chloride (m.w. 76.6)and 80.0 g sodium chloride (m.w. 58.5), and adding dH₂O to a finalvolume of 1 liter. A 1× working stock of DPBS is prepared by mixing 100ml 10× DPBS with 900 ml dH₂O.

Following the DPBS wash, slides are placed in an antigen unmaskingsolution (0.1M sodium citrate, pH 6.0), placed in a pressure-cooker, andheated in a microwave oven at 600 W twice for 15 minutes each. Theantigen unmasking solution is made by mixing 810 ml of 0.1 M sodiumcitrate.2H₂O (m.w. 294.12; 29.4 g/1000 ml) with 190 ml of 0.1 M citricacid.H₂O (m.w. 210.14; 5.252 g/250 ml), for a final volume of 1 liter.Following the microwave treatment, the slides are left in the antigenunmasking solution for an additional 20 minutes at ambient temperature.

Prior to immunohistochemical staining of freshly frozen tissue biopsysections on glass slide, the fresh frozen biopsy sections are fixed incold-acetone for 5 minutes, air-dried for 30 minutes and incubated twicein DPBS for 5 minutes each.

The slides containing formalin-fixed or acetone-mixed tissue sectionsare next incubated for 20 minutes in a 1:20 dilution of non-immune horseserum, (Cat. No. S-2000), available from Vector Laboratories,Burlingame, Calif. This is followed by incubation in appropriatedilutions of the anti-JI-31 mouse monoclonal antibody from 60 minutes toovernight. The appropriate dilution of the antibody is dependent on anumber of factors, including the concentration of the antibody beingused, and may be determined empirically by techniques known to oneskilled in the art.

Following incubation with the primary antibody, the slides are washed inDPBS four times, for two minutes each wash. The slides are thenincubated in appropriate dilutions of biotinylated antibody for 30minutes. One biotinylated antibody suitable for use with the anti-JI-31mouse monoclonal antibody is a 1:200 dilution of biotinylated horseanti-mouse polyclonal antibodies (IgG, H+L), available from VectorLaboratories, Cat. No. BA-2000. This is followed by another series offour 2-minute washes in DPBS.

The slides are next incubated for 30 minutes inavidin-biotin-horseradish peroxidase complex (ABC), available in a kitfrom Vector Laboratories, Cat. No. PK-6100. In this procedure, apreformed Avidin (A):Biotinylated enzyme (B) complex is used withbiotinylated antibodies. 25 μl each of the “A”]and “B” reagents providedin the Vector kit are added to 2.45 ml DPBS for a final volume of 2.5ml. Following incubation, the slides are washed 4 times for 2 minuteseach in DPBS.

The final steps in the immunostaining procedure is to expose the slideswith bound antibody-ABC to a chromagen substrate for the ABC enzyme. Onechromagen suitable for use with horseradish peroxidase isdiaminobenzidine (DAB). Slides prepared as above are incubated for 10minutes in a DAB solution made by combining 6.0 mg DAB (Sigma Chemical,Cat. No. D-5637) with 9.9 ml DPBS and 0.1 ml 3% H₂O₂, for a final volumeof 10.0 ml. The slides are then washed five time for 2 minutes each inwater, followed by a 1 to 2 minute incubation in Harris hematoxylin, tocounterstain the tissue, and another series of five 2-minute washes inwater.

Finally, the slides are incubated two-times in 95% ethanol for 3 minuteseach incubation, followed by two 3-minute incubations in absolutealcohol, and ending with two 3-minute incubations in xylene. The slidesare then provided with a coverslip in permount mounting medium.

Although one particular protocol has been described for use inimmunohistochemical staining tissue samples with the anti-JI-31 mousemonoclonal antibody, other anti-JI-31 antibodies and immunostainingtechniques may also be employed. For example, in addition to peroxidaseconjugates, alkaline phosphatase conjugates may be used. Other suitableimmunohistochemical procedures are apparent to the skilled practitioner.

Immunolabeling for Fluorescence Microcopy

Fluorescence microscopy may be used to detect JI-31 in astrocytes orhigh grade astrocytomas in cell culture or in tissue sections (Singh,supra, Dendron 1: 91-108).

Briefly, for cultured cells, coverslips bearing cells are fixed ineither methanol (5 minutes at −20° C.) or 4% paraformaldehyde (30minutes at room temperature). The coverslips are then washed inphosphate buffered saline (PBS), pH 7.4, containing 0.05% Tween-20(PBS-T).

The coverslips are incubated in 30% normal goat serum in PBS for 30minutes to prevent non-specific binding. Anti-JI-31 antibody is added asthe primary antibody and incubated overnight at 4° C. For example,antibody JI-31 ascites fluid may be applied at a 1:500 dilution in PBS;also, normal mouse serum (NMS; Sigma) may be applied to some coverslipsas a control. Following a wash in PBS-T, an appropriate secondaryantibody (for example, a 1:100 dilution in PBS of goat anti-mouse IgGconjugated to fluorescein isothiocyanate (FITC; Sigma)) is added and thecoverslips incubated for one hour at room temperature in the dark.

Optionally, the cells are double-labeled by first washing the coverslipsagain in PBS-T, followed by the application of a second primary antibodyand incubation overnight at 4° C. Suitable antibodies fordouble-labeling included rabbit anti-cow glial fibrillary acidic protein(GFAP; Dimension Laboratories; 1:500 dilution in PBS), and its control,normal rabbit serum (NRS; Sigma); alternatively, Mab Vim 13.2 (amonoclonal antibody to vimentin; Sigma) and its NMS control may be used,also at a 1:500 dilution. The coverslips are washed in PBS-T and goatanti-rabbit IgG-TRITC (Sigma) or goat anti-mouse IgM-TRITC (Sigma) isapplied at a 1:100 dilution in PBS. The coverslips are then incubated inthe dark for 1 hour at room temperature

Following incubation, the coverslips are given a final wash in PBS-T,mounted in glycerol/□-phenylenediamine and viewed by fluorescencemicroscopy and laser scanning confocal microscopy. Those cellsexpressing JI-31 antigen, and thus incorporating anti-JI-31 antibody andFITC-labeled secondary antibody, will fluoresce at a particularwavelength when excited, while any low grade astrocytoma cells presentin the sample will not fluoresce at that wavelength, as no anti-JI-31antibody/FITC-labeled secondary antibody is incorporated, allowing thelow grade astrocytoma cells to be distinguished from the JI-31containing normal astrocytes and high grade astrocytomas. Where doublelabeling is used, the second fluorescent dye is selected so as tofluoresce at a different wavelength (or in response to a differentexcitation wavelength).

For immunofluorescence microscopy of tissue sections, the tissuesections are fixed in either 4% paraformaldehyde or methanol andsectioned using a cryostat or vibratome. Imprints on coverslips fromfresh specimens may also be similarly fixed. Sections and imprints areimmunostained as described above.

Other fluorescent dyes, known in the art, may also be used in thepractice of the present invention.

Immunoelectron Microscopy

Immunoelectron microscopy can also be used to detect GFAP and JI-31proteins at the ultrastructural level in biopsy samples (Singh, supra,Dendron 1: 91-108). Briefly, specimens are fixed in 1.25% glutaraldehydeand embedded in LR white resin by routine methods. Ultrathin sectionsare etched with sodium metaperiodate and double-immunolabeled withanti-JI-31 antibody (preferably, monoclonal anti-JI-31 antibody) andanti-GFAP antibody as primary antibodies. The presence of JI-31 and/orGFAP is detected by the presence of the primary antibodies, asdetermined using secondary antibodies labeled with gold particles ofdistinct size (for example, 5 nm for anti-JI-31 antibody and 10 nm foranti-GFAP antibody). Thus, cells displaying both 5 nm and 10 nmparticles (those expressing both JI-31 and GFAP; i.e., normal astrocytesand high grade astrocytomas) may be distinguished from astrocytesdisplaying only 10 nm particles (those expressing GFAP but not JI-31;i.e., low grade astrocytoma cells).

SDS-PAGE and Immunoblotting

The presence (or absence) of JI-31 in cells, both cultured and in tissuespecimens, may be evaluated using standard sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and western blotprotocols, which also allow the determination of the molecular weight ofJI-31 protein (Singh, supra, Dendron 1: 91-108).

Protein extracts from cultured cells (or tissue homogenates) areelectrophoresed under reducing conditions in a discontinuous gel, thentransferred to nitrocellulose or other suitable membrane byelectrophoretic or other transfer. The membranes are then treated withanti-JI-31 primary antibodies and labeled secondary antibodies, usingstandard western blot procedures.

In one embodiment, suspected astrocytoma cells can be selectivelydissected from tissue samples using, for example, the lasermicrodissection technique of PALM® (P.A.L.M. Microlaser Technologies AG,Germany). Protein extracted from these cells can then be subjected toSDS-PAGE/western analysis to evaluate the presence or absence of JI-31.

Kits for the Detection of JI-31

For diagnosis of low grade astrocytomas, a kit for the detection ofJI-31 in tissue specimens and/or cultured cells is used. The kitcomprises anti-JI-31 antibody, optionally JI-31 antigen for use as acontrol, and a means for detecting the complexing of the antibody withJI-31 antigen in the cultured cells or tissue specimen.

The kit detects the JI-31 antigen, when present, with the anti-JI-31antibody. The complexing immunoreaction is detected by staining,fluorescence, immunoprecipitation or by any other means used in the artand suitable for these purposes.

In addition to the above, the kits may also comprise control compounds,anti-antibodies, protein A/G, anti-GFAP antibodies and the like for usein double-labeling, etc., suitable for conducting the different assaysreferred to above.

Having generally described the invention, the same will be more readilyunderstood through reference to the following example, which is providedby way of illustration, and is not intended to be limiting of thepresent invention.

EXAMPLE 1 Localization of JI-31 Antigen and Glial Fibrillary AcidicProtein (GFAP) in Formalin-Fixed/Paraffin-Embedded Tissue Sections usingImmunohistochemical Staining

Formalin-fixed and paraffin-embedded tissue sections of a low gradeastrocytoma were prepared and stained, as described above. Briefly, thetissue sections were treated as follows:

-   -   1. Slides of the tissue sections were warmed overnight at 60° C.    -   2. The slides were incubated in Histoclear for 5 minutes;        repeated once.    -   3. The slides were incubated in absolute ethanol for 3 minutes;        repeated once.    -   4. The slides were incubated in 95% ethanol for 3 minutes;        repeated once.    -   5. The slides were incubated in H₂O₂:methanol for 20 minutes.    -   6. The slides were washed in DPBS for 5 minutes.    -   7. The slides were treated in antigen unmasking solution (0.1 M        sodium citrate buffer, pH 6.0) in a pressure-cooker and heated        in a microwave oven at 600 W twice for 15 minutes each.    -   8. The slides were left in the antigen unmasking solution for an        additional 20 minutes at ambient tempterature.    -   9. The slides were incubated in non-immune horse serum for 20        minutes.    -   10. The slides were incubated overnight in a humidified chamber        in either a 1:1,000 dilution of anti-JI-31 monoclonal mouse        antibody or a 1:50 dilution of 113 μg/ml monoclonal mouse        anti-human GFAP antibody (Cat. No. M 0761, Dako Corporation,        Carpenteria, Calif.).    -   11. The slides were washed in DPBS for 2 minutes; repeated three        times.    -   12. The slides were incubated in a 1:200 dilution of        biotinylated horse anti-mouse polyclonal antibodies for 30        minutes.    -   13. The slides were washed in DPBS for 2 minutes; repeated three        times.    -   14. The slides were incubated in avidin-biotin-horseradish        peroxidase complex (Vector Labs PK-6100 Elite) for 30 minutes.    -   15. The slides were washed in DPBS for 2 minutes; repeated three        times.    -   16. The slides were incubated for 10 minutes in        diaminobenzidine.    -   17. The slides were washed in water for two minutes; repeated        four times.    -   18. The slides were incubated in Harris hematoxylin for 2        minutes.    -   19. The slides were washed in water for two minutes; repeated        four times.    -   20. The slides were incubated in 95% alcohol for 3 minutes;        repeat once.    -   21. The slides were incubated in absolute alcohol for 3 minutes;        repeat once.    -   22. The slides were incubated in xylene for 3 minutes; repeat        once.

The slides were then prepared in permount mounting medium and examinedmicroscopically at 200× magnification. Photomicrographs of the stainedcells are shown in FIG. 1, A through D.

Cells stained with anti-JI-31 antibody are shown in FIGS. 1B and 1D,while cells stained with anti-GFAP antibody are shown in FIGS. 1A and1C. As shown in FIGS. 1A and 1B, mouse monoclonal anti-GFAP antibody oranti-JI-31 antibody shows strong reactivity with normal astrocytes asshown by the astrocytes stained in brown (for example, by the arrow inFIGS. 1A (GFAP) and 1B (JI-31)).

The low grade (grade 2) astrocytoma cells, however, show no reactivitywith anti-JI-31 antibody (note the absence of brown staining, forexample, near the arrow, FIG. 1D), whereas the surrounding normalastrocytes were positive (note the presence of brown staining near the*, FIG. 1D). The blue-stained nuclei from the Harris hematoxylincounterstain indicate the presence of cells. In contrast, anti-GFAPantibody reacted with both the normal astrocytes and the grade 2astrocytoma cells (see, for example, the brown-stained cells near thearrow, FIG. 1C).

Thus, as demonstrated above, anti-JI-31 antibody can be used to quicklyand efficiently diagnose the presence of low grade astrocytoma cells ina patient, allowing for improved screening and treatment of patientsexperiencing neurological cancer.

All patents, patent applications, and other publications mentioned inthis specification are incorporated herein in their entireties byreference.

While this invention has been described in detail with reference to acertain embodiments, it should be appreciated that the present inventionis not limited to those precise embodiments.

Rather, in view of the present disclosure which describes the currentbest mode for practicing the invention, many modifications andvariations would present themselves to those of skill in the art withoutdeparting from the scope and spirit of this invention. The scope of theinvention is, therefore, indicated by the following claims rather thanby the foregoing description. All changes, modifications, and variationscoming within the meaning and range of equivalency of the claims are tobe considered within their scope.

1. A method for identifying low grade astrocytoma cells in a biologicalsample, comprising the steps of: preparing slides from a biologicalsample; serially incubating said biological sample in at least ethanoland hydrogren peroxide; heating said slides in an unmasking solutionincluding a mixture of at least sodium citrate and citric acid;incubating the biological sample with anti-JI-31 antibody underconditions in which the antibody binds to JI-31 antigen in the sample;contacting the biological sample with a secondary antibody selected soas to form an immune complex with the anti-JI-31 antibody bound to JI-31antigen; incubating the immune complex with a conjugate; exposing theconjugated immune complex with a chromagen; wherein after eachincubation the slide is washed at least four times; and detecting cellsexpressing the JI-31 antigen by detecting the immune complex; anddetecting astrocytes in the sample in which the immune complex is notpresent, to thereby identify low-grade astrocytoma cells.
 2. The methodof claim 1, wherein the secondary antibody is biotinylated.
 3. Themethod of claim 2, wherein the immune complex is detected by contactingthe sample with an avidin.-peroxidase conjugate, followed by a chromagensuitable for use as a peroxidase substrate.
 4. The method of claim 1,wherein the secondary antibody is labeled with a fluorescent dye.
 5. Themethod of claim 1, wherein the secondary antibody is labeled with ametal particle.
 6. The method of claim 1, wherein each wash includes atleast an 0.05% solution of Tween-20.
 7. The method of claim 1, whereinthe biological sample is fixed in one of either paraformaldehyde ormethanol.
 8. The method of claim 1, wherein the sample is furtherstained with a non-cell specific stain.
 9. The method of claim 1,wherein the anti-JI-31 antibody is a monoclonal antibody.
 10. The methodof claim 8, wherein the anti-JI-31 antibody is produced by the hybridomacell line JI-31, ATCC designation number CRL-2253.
 11. The method ofclaim 1, further comprising the step of contacting the sample withanti-GFAP antibody under conditions in which the anti-Glial FibrillaryAcidic Protein antibody binds to GFAP antigen in the sample.
 12. Themethod of claim 6, wherein the anti-Glial Fibrillary Acidic Proteinantibody is labeled with a second fluorescent dye selected to fluoresceat a distinct wavelength.